Project Summary Mycobacteria are a diverse group of high G+C Gram-positive bacteria, with an extremely complex cell envelope consisting of the mycolyl-arabinogalactan-peptidoglycan complex with a plethora of associated lipid and glycolipids. This hallmark of the mycobacteria is an important determinant of the biology of these organisms and has been the focus of much of the research on M. tuberculosis and other mycobacteria. The work we propose here on peptidoglycan-protein ligation would open up new avenues of study on the biogenesis and organization of the mycobacterial cell envelope. We are focusing on highly pathogenic M. tuberculosis, which is still a major health care issue in the 21st century, and infects approximately one-third of the world's population. This work could have an important impact on various aspects of M. tuberculosis biology including pathogenesis, drug, and vaccine development. We also plan to analyze the faster-growing, lesser pathogenic environmental species M. smegmatis and M. abscessus. While M. smegmatis is rarely a pathogen, it has a broader environmental reservoir than M. tuberculosis and has a larger genome, and is the workhorse species for mycobacterial genetics and physiology. M. abscessus is an important emerging environmental non- tubercular mycobacteria (NTM) that is a growing problem in elderly populations and in Cystic Fibrosis patients. Treatment of M. abscessus infections is difficult due to inherent resistance to antibiotics, and not much is known about this species compared to M. tuberculosis or M. smegmatis. Hence, our investigations into this species would shed new light on the biology of NTM.